Platelets are a component of blood comprised of anucleate megakaryocyte fragments that circulate in the blood for about 10 days. When separated as a component of whole blood, platelets are routinely concentrated, re-suspended in plasma and/or platelet additive solutions, leukoreduced by passage through a filtration device and stored in platelet storage bags which are kept on flatbed agitators for 5 to 7 days at a temperature of 22° C. The relative DNA content in whole blood platelets (WBP) or leukoreduced/apheresis platelets varies considerably.
Microbial contamination of blood transfusion products is a major medical problem. Blood banks are faced with a great challenge in testing each platelet bag for microbial contamination prior to release for infusion into a patient. Currently contaminated platelets are often infused into patients, and the physician is notified subsequently that the platelets were contaminated as the culture results become available. Under the American Association of Blood Banks (A.A.B.B.) standard 5.1.5.1, blood banks or transfusion services are instructed to have methods to limit and detect bacterial contamination in all platelet concentrates. Nucleic Acid Testing (NAT) technology would allow testing of bacterially contaminated units to be detected rapidly after the first day of storage, thus ensuring a safe transfusion by eliminating the possibility of contaminated platelets.
With the successful implementation of stringent control measures, the estimated risk of infection by well known viral pathogens such as HIV and HCV has fallen below 1 per 1 to 2 million transfusion units. This was achieved in part through the use of nucleic acid based testing performed on pooled products composed of 16 to 24 units. In contrast, the risk of transfusion with bacterially contaminated platelets may be as a high as 1 in 1,000 units, with perhaps 10% to 25% of such incidents resulting in adverse effects on patients. A recent study on the microbiological safety of transfusions concluded that a rapid test for microbial contamination in platelets with a detection limit of 103 or 104 organisms/ml is a desirable target.
Vogelstein and Gillespie described the purification of DNA using glass as a DNA binding surface in the presence of high concentrations of chaotropic salts, for example, GuSCN, GuHCl, NaI, or NaClO4. The binding of sequence specific DNA probes to glass microscope slides for use in DNA microarrays was also previously described. The slides were not, however, used for capture of genomic DNA from samples. One advantage of the flat glass microscope slides is the reproducibility of the glass product. Glass microscope slides are made by Erie Scientific (Portsmouth, N.H.) from soda lime glass. Thin glass sheets are drawn from the molten glass using the “electroverre” process and the material is known as “Swiss glass.” Glass microscope slides are cut from large thin (1 mm thick) sheets. Glass slides have been used for decades in medical diagnostics and the material is very uniform.
U.S. Pat. No. 5,234,809 describes a one-step process for purification of nucleic acids from complex material such as body fluids or other biological starting materials. In the method, a process is described for isolating nucleic acids from starting material comprising mixing the starting material, a chaotropic substance, and a nucleic acid solid phase such as glass cuvettes, separating the solid phase with the nucleic acid bound to it from the liquid, and washing the solid phase nucleic acid complexes. However, it is well known that such a one-step process often cannot be used to purify a significant amount of the total nucleic acids available in the starting mixture. This can be demonstrated using even known quantities of nucleic acids added to a complex biological fluid—plasma—prior to purification over a well understood commercial DNA purification kit using processes such as those described in U.S. Pat. No. 5,234,809.
For extraction of nucleic acids from blood or blood products, proteolysis of the sample is commonly carried out prior to purification of the DNA. Enzymes such as proteinase K, lysostaphin, or other similar enzymes that are either expressed in organisms such as Escherichia coli or Pichia pastoris and purified, or purified from other sources, can be used for proteolysis.
NAT is a powerful analytical tool for determining the presence of genetic material (DNA or RNA) in biological samples. For example, polymerase chain reaction (PCR) can be used to detect trace microbial contamination in sterile systems and to find pathogenic gene sequences in the human cells. In blood banks, NAT is commonly used to detect the presence of viral contamination (HIV, HBV, HCV) in blood products.
It has been previously shown using a NAT protocol that the sampling of pooled platelets after one day of storage affords the accurate detection of most bacterial species in spiked platelet concentrates (PCs) at detection levels that equal or exceed the culture systems currently in use for bacterial testing of PCs. The NAT protocol provides an inherent advantage in providing results within a short amount of time in a closed sterile device that would allow for the clinical determination of the level of microbial contamination within a sample.